Increase in the community circulation of ciprofloxacin-resistant Escherichia coli despite reduction in antibiotic prescriptions

Background Community circulating gut microbiota is the main reservoir for uropathogenic Escherichia coli, including those resistant to antibiotics. Ciprofloxacin had been the primary antibiotic prescribed for urinary tract infections, but its broad use has been discouraged and steadily declined since 2015. How this change in prescriptions affected the community circulation of ciprofloxacin-resistant E. coli is unknown. Methods We determined the frequency of isolation and other characteristics of E. coli resistant to ciprofloxacin in 515 and 1604 E. coli-positive fecal samples collected in 2015 and 2021, respectively. The samples were obtained from non-antibiotic-taking women of age 50+ receiving care in the Kaiser Permanente Washington healthcare system. Results Here we show that despite a nearly three-fold drop in the prescription of ciprofloxacin between 2015 and 2021, the rates of gut carriage of ciprofloxacin-resistant E. coli increased from 14.2 % to 19.8% (P = .004). This is driven by a significant increase of isolates from the pandemic multi-drug resistant clonal group ST1193 (1.7% to 4.2%; P = .009) and isolates with relatively few ciprofloxacin-resistance determining chromosomal mutations (2.3% to 7.4%; P = .00003). Though prevalence of isolates with the plasmid-associated ciprofloxacin resistance dropped (59.0% to 30.9%; P = 2.7E-06), the isolates co-resistance to third generation cephalosporins has increased from 14.1% to 31.5% (P = .002). Conclusions Despite reduction in ciprofloxacin prescriptions, community circulation of the resistant uropathogenic E. coli increased with a rise of co-resistance to third generation cephalosporins. Thus, to reduce the rates of urinary tract infections refractory to antibiotic treatment, greater focus should be on controlling the resistant bacteria in gut microbiota.

BLA and VF determinants, number and type of QRDR mutations in GyrA and ParC and ciprofloxacin MIC.

Study design and participants
Both 2015 and 2021 studies were approved by the Kaiser Permanente Washington (KPWA) Research Institute Institutional Review Board. Study protocols included waivers of consent to identify potential participants. Risks and benefits of the research were explained to participants in a mailed invite letter and consent information sheet. Participants provided informed consent for participation by sending a biological sample back in the mail. The 2015 study was carried put between July, 2015 and December, 2016, the 2021 study between May-November 2021. Initially, both studies had a crosssectional random sample of 6,000 women ≥18 years old (2015) and 6,750 women ≥50 years old (2021), enrolled at KPWA for at least one year and neither diagnosed for UTI nor treated with antibiotics in the 12 months prior to the sample pull date in April 30, 2015 (2015 study) and April 29, 2021 (2021 study) were pre-selected. The recruitment of participants from this sample was carried out as follows: every week a random subgroup of 100-300 selectees were contacted by mail with an explanation of the project and instructions for their fecal samples to be obtained by using a self-collection kit. For 2015 study, the mailing lasted for 6 months, with 3,367 kits mailed out and 1,032 kits returned (31% response rate). Out of those, 575 kits were from 50+ yo women and thus were included in the current study. For 2021 study, 6400 kits were mailed between May and November 2021, with 1778 kits with fecal samples returned (28% response rate). The mailings were stopped early when the intended sample size of 1,000 (2015) and 1600 (2021) samples was reached.

Sample processing
Fecal samples were collected using the FecalSwab™ Sample Collection and Preservation System for Enteric Bacteria by Copan Diagnostic Inc. (Carlsbad, CA, USA). Samples were visually assessed for the quality of fecal matter before being plated on four types of agar. (1) Pre-poured HardyCHROM™ UTI agar plates (Hardy Diagnostic, USA) were used for non-antibiotic plating of E. coli. The proprietary composition of these plates allows for the differential detection of uropathogenic microorganisms. (2)(3)(4) For plating on ciprofloxacin, plates containing ciprofloxacin at 0.5, 2 or 10 mg/L were prepared using HiChromeTM UTI Agar (HiMedia Laboratories Pvt, Ltd., India). The rest of the sample was split into two tubes (with and without 10% glycerol) and stored at -80oC. The plates were incubated for 16-20 hours at 37°C, and up to 30 single colonies (SCs) that were morphologically identified as potential E. coli (magenta, pink, or opaque color) were cultured, saved, and further tested. All single colonies were tested for (a) growth on ciprofloxacin-supplemented agar and (b) clonality based on sequencing of four loci (see below). Fluoroquinolone-resistant E. coli (FQREC) were defined as single isolates that grew on plates with at least 0.5 mg/L of ciprofloxacin. FQREC were further tested for ciprofloxacin MIC and susceptibility to third-generation cephalosporins (3GC) (see below). Additionally, subsets of FQREC were tested for the presence of PMQR, bla, and VF loci (see below).
Identification of FQREC clonality and number of QRDR mutations E. coli clonality was determined by CH typing based on fumC/fimH sequencing 1 , presence of QRDR mutations was determined by sequencing of gyrA and parC 2 . All reactions were carried out by 2-step colony PCR. Briefly, a single FQREC colony was resuspended in 50 µL of sterile water and heated at 98°C for 10 min. Primary PCR reactions were set up in a 15 µL volume using DreamTaq Mastermix (Thermofisher, USA), supplemented with 0.5 µM forward and reverse primers, and 1.5 µL of the boiled colony template. Primary PCR was run for 30 cycles under the manufacturer's recommended conditions. Subsequently, 1 µL of the PCR1 product was used for an additional PCR reaction, using nested forward and reverse primers supplemented with T7 and T7-Term tails, respectively. The nested PCR was run for 15 cycles under the same conditions, aiming to obtain a highly specific single band with T7-tailed primers suitable for downstream sequencing. The primer sequences can be found in Supplemental Table S7.

Identification of 3GCR FQREC
To identify FQREC that were non-susceptible to third-generation cephalosporins (3GCR), the isolates were tested on plates containing cefoxitin at 4 and 8 mg/L (CEF-4 and CEF-8), ceftazidime at 8 and 16 mg/L (CAZ-8 and CAZ-16), and HardyCHROM ESBL HDx (Hardy Diagnostics, USA, containing ceftazidime and cefpodoxime). The ESBL vs. non-ESBL type of 3GC-non-susceptibility was determined based on growth on the aforementioned plates and the difference in diameter growth inhibition by ceftazidime and ceftazidime/clavulanate, as described in CLSI 3 .

Testing antibiotic resistance of E. coli isolates
Resistance of E. coli isolates to a panel of 12 antibiotics was tested for 383 FQREC (from 361 samples) using Kirby-Bauer disk diffusion method as described in CLSI manual 3 . The antibiotics disks were purchased from Hardy Diagnostics (Santa Maria, CA, USA) and are as follows: AMP, ampicillin, AMC, amoxicillin/clavulanate, CZ, cefazolin, IMI, imipenem, T/S, trimethoprim/sulfamethoxazole, TET, tetracycline, FOS, fosfomycin, NIT, nitrofurantoin. For a random set of 255 isolates testing was repeated.

Determining presence of Plasmid-Mediated Quinolone Resistance loci (PMQR)
The PMQR (or TMQR) loci were identified using 8-loci multiplex described in Ciesielczuk et al. 4 with addition of ninth locus qnrVC from Kraychete et al 5 . The primers are listed in Supplemental Table 1. For testing, single colonies of FQREC isolates were resuspended in 50 µL sterile water, boiled 10 min, and 1.5 µL was used as template in a 20 µL PCR reaction with 0.5 µM primers supplied. In-house strains from the Sokurenko Lab were used as positive controls (except for qnrVC, for which there was no positive control). The presence of weak bands in the multiplex was confirmed or refuted in a singleplex test using appropriate primers.

Determining absence/presence of Virulence Factors (VF)
The presence of uropathogenic virulence factors (gadA/B 6 , iha 7 , ireA 8 , sat 9 , vat 10 and senB 11 ) was determined by the presence of PCR products after an amplification reaction using primers listed in Supplemental Table 1. Reactions were carried out using boiled bacteria as template, as described above. In-house strains from the Sokurenko Lab were used as positive controls.
Determining absence/presence of beta-lactamase loci (BLA) The presence of major determinants of beta-lactamases was detected using five multiplexes described in Dallenne et al. 12 Primers are listed in Supplemental Table 1. Additionally, primers specific for blaNDM were added to Multiplex III. Reactions were carried out using boiled bacteria as template as described above. In-house strains from the Sokurenko Lab were used as positive controls. The presence of weak bands in multiplex was confirmed or refuted in singleplex test using appropriate primers.  Total VF+ 108 None found 50 a The tested isolates belonged to 25 clonal groups including ST131-H30 (37), ST131-H41 (10)   Overall, in 84% of tested 3GCR isolates at least one BLA determinant was found, with 24.0% of those carrying more than one determinant; in 16% cases no genes potentially responsible for the ESBL phenotype could be identified.